The post-menopausal period in women's life is challenging for their health and is associated with increased risk of musculoskeletal impairments. Women undergoing the menopausal transition experience both, age as well as gonadal hormone related changes in physiological functions. As a result, accelerated loss in bone mass density (BMD), muscle mass and muscle strength occur in women undergoing menopause, unlike men of the same age. The common musculoskeletal disorders among postmenopausal women (PMW) are osteoporosis, osteoarthritis (OA). Postmenopausal women with osteoarthritis have a 20% increased risk of fracture and experience 25% more falls than those without osteoarthritis (Prieto-Alhambra et al. 2013).
The risk is high in women as compared to men and increases sharply after 50 years of age. The quality of life is greatly impaired in persons with sever osteoporosis. It is known to affect >50% of women and 30% men over the age of 50 years. The reduction in bone strength associated with this disease markedly increases the risk of fractures, and the consequent pain and loss of function have adverse effect on the quality of life. For fractures and osteoarthritis there are no drugs in the market.
Most of the pharmacological agents available for clinical use for osteoporosis are calcium, vitamin D and its analogue, calcitonin, bisphosphonates, raloxifene, hormone replacement therapy (HRT) etc. act by decreasing the rate of bone resorption, thereby slowing the rate of bone loss. Timely administration of such antiresorptive agents prevents bone loss. Hormone replacement therapy, though effective in preventing bone loss following ovariectomy or menopause in women, is associated with increased risk of endometrial hyperplasia and carcinoma [Grady, D. Grebretsadik, T. Ernestwr, V. Petitti, D. Gynecol. 85, 304-313 (1995), Beresford S. A. Weiss, N. S. Voigt, L. F. McKnight, B. Lancet 349, 458-461 (1997)], breast cancer [Riggs, L. Hartmann, L. C. J. Med. 348, 618-629, (2003)], and thromboembolic diseases [Delmas, P. D. Lancet 359, 2018-2026 (2002)]. Bisphosphonates are poorly absorbed and may cause gastrointestinal irritation, diarrhoea and constipation. Raloxifene has been reported to increase incidence of hot flashes, deep vein thrombosis, pulmonary embolism and leg cramps [Clemett, D., Spencer, C. M. Drugs 60, 380-409 (2000)]. Though anti-resorptive therapies are available and are in use, anabolic/osteogenic therapies are limited to PTH, NaF and strontium which are also accompanied by undesirable side effects (Rosen and Bilezikian 2001, Lane and Kelman 2003). Thus there is a need for the development of agents that could increase bone mass and strength, thereby reducing the risk of osteoporotic fracture more than the currently available anti-resorptive agents.
The importance of traditional medicine as a source of primary health care was first officially recognized by the World Health Organization (WHO) in 1976 by globally addressing its Traditional Medicine Programme. In traditional medicine, there are many natural crude drugs that have the potential to treat bone diseases. However, not much laboratory work has been reported evaluating their possible development and use, except ipriflavone, a natural product derivative, which has been used clinically for such indications [Fujita, T., Yoshikawa, S., Ono, K., Inoue, T., Orimo, H. J. Clin. Exp. Med. 138, 113-141 (1986), Passeri, M., Biondi, M., Costi, D., Bufalino, L., Castiglione, g. N., DiPeppe, C., Abate, G. Bone Miner. 19 (Suppl. 1), S57-62 (1992)]. It is believed that herbal medicines are easily available, less expensive, and safer than chemically synthesized drugs. In India Ayurvedic medicine emerged during the rise of the philosophies of the Upanishads, Buddhism, and other schools of thought in India. Herbs played an important role in Ayurvedic medicine. In our program search for natural osteogenic plant, n-butanol soluble fraction of ethanol extract of Dalbergia sissoo aerial part which is renewable source exhibited osteogenic activity in our test model. Thus, the plant extract might possess bioactive ingredients that could promote bone formation. The effects on osteoporosis and total osteo-health and related disorders and has not been explored.
There is, thus, an urgent need to discover and develop a promising herbal product exhibiting promising bone anabolic or for bone forming activity in experimental animals and human beings for osteoporosis and osteoarthritis. The Spinacia oleracea is a fit case to study and explore its true potential with respect to its bone forming response of its extract, fraction and pure biologically active marker components. The experiments have shown that its extract exhibit promising bone forming activity.
S. oleracea L. commonly known as Spinacea, is an edible flowering plant in the family of Amaranthaceae. It is an erect herb about 30-60 cm height, native to South-West Asia and cultivated throughout world as vegetables. Fresh plants are boiled and eaten to increase appetite and as vitamin supplement. Although less in calorific value, spinacea is recommended as the best source of iron, vitamins, minerals and mineral salts, 100 g of spinacea provide more than 20% of the daily Value of vitamin A, C and vitamin K, magnesium, manganese, folate and iron and 10-19% of the daily Value of B vitamins, riboflavin and vitamin B6, vitamin E, calcium, potassium especially when fresh or quickly boiled. [The wealth of India. Vol 5. New Delhi: National Institute of Science, Communication & Information Resources (CSIR), 2004: pp 146-7].
Ayurveda and Unani system of medicine describe the plant as carminative, laxative, alexipharmic, leaves are emollient, antipyretic, diuretic, maturant, laxative, digestible, anthelmentic useful in diseases of blood and brain, asthma, leprosy, biliousness, urinary concretion, leucorrhoea, inflammation of the lungs and the bowels, sore throat, pain in joints, thirst, lumbago, cold and sneezing, sore eye, ring worm scabies, leucoderma, soalding urine, arrest vomiting, biliousness, flatulence and in the treatment of febrile conditions. Seeds are also used in the treatment of difficulty in breathing, inflammation of the liver and jaundice urinary calculi.
Different parts of spinacea and extracts have been demonstrated to exert numerous beneficial effects such as anti-oxidative, hepato-protective, anti-inflammatory, anticancer, chemo-protective, central nervous system protection and anti-aging functions. Consumption of polyphenols rich fresh vegetables has been associated with a reduced risk of oxidative stress-induced disease. Water extracts of spinacea leaves demonstrated their biological activity in both in vitro and in vivo systems [Lomnitski, L., Bergman, M., Nyska, A., Ben-Shaul, V., Grossman, S. Nutr Cancer 2003, 46, 222-231]. Maximum antioxidant effect was exhibited in the ethanol fraction (11.55%) followed by ethyl acetate fraction (10.51%) n-hexane fraction (4.12%) antioxidant activity in dose-dependent way in DPPH radical scavenging assey [Arfan, M., Gul, S., Usman, R., Khan, A., Rauf, A., Muhammad, N., Shah, S. U. A., Khan, A., Ali, M. Academic J of Plant Sci. 2013, 6, 3]. Recovered antioxidant flavonoids, in particular spinacetin and petuletin, content from a crude spinacea extract by the elimination of a major part of non-phenolic components by adsorption procedure exhibited significantly increased antioxidant efficiency in nitro blue tetrazolium (NBT) assay and the diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assey [Aehle, E., Raynaud-Le Grandic, S., Ralainirina, R., Baltora-Rosset, S., Mesnard, F., Prouillet, C., Mazière, J. C., Fliniaux, M. A., Food Chem. 2004, 86, 579-585].
A water-soluble natural antioxidant mixture (NAO) isolated from spinacea leaves specifically inhibits the lipoxygenase enzyme [Lomnitski, L., Bergman, M., Nyska, A., Ben-Shaul, V., Grossman, S., Nutr Cancer 2003, 46, 222-231]. Spinacea extracts has beneficial effects on body weight and serum lipids in obese postmenopausal and premenopausal women and in men. Ecdysone is produced by spinacea and spinacea extracts containing ecdysone decreased body fat load, thereby reducing secretion of proinflammatory cytokines by visceral adipocytes and oxidative stress [Seidlova-Wuttke, D., Jarry, H., Wuttke, W., Horm. Mol. Biol. Clin. Invest. 2013, 16, 35-45].
Supplementation of plant extract, extracted with 50% methanol to mice for 15 days showed radioprotective effect against gamma radiation-induced oxidative stress the check of radiation-induced damage in mice liver at the optimum dose of 1100 mg/kg body wt./day [Bhatia, A. L., Jain, M., Phytomedicine 2004, 11, 607-615]. Pretreatment with alcoholic extract prior to the administration of CCl4, at the doses of 100 and 200 mg/kg/day, for 7 days, significantly restored to all the serum and liver parameters, serum-marker enzymes like GGT, AST, ALT, LDH, SDH, GDH, ALP, near to the normal levels [Gupta, R., Singh, D. Pharmacologyonline 2006, 3, 267-278; Al-Dosari, M. S. Clinical and Experimental Medical Journal 2010, 4, 129-140]. Additionally, n-butanol fraction of Spinacia oleracea showed significant protection against CCl4-induced hepatotoxicity. The presence of 20-hydroxyecdysone (20-HE) in the n-butanol fraction of Spinacia oleracea further confirmed by HPTLC analysis may be responsible the effects [Jain, N. K., Singhai, A. K., Asian Pac. J. Trop. Biomed., 2012, 2, S232.].
Spinacea extract and natural antioxidant mixture (NAO) isolated from spinacea have shown anticancer effect on various types of cancer such as ovarian, lung, prostatic, breast and colon. Spinacea showed antiproliferative activities in-vitro using HepG(2) human liver cancer cells [Chu, Y.-F., Sun, J., Wu, X., Liu, R. H., J Agric. Food Chem., 2002, 50, 6910-6916].
Spinacea extract NAO was tested both in-vivo and in-vitro in cell lines DU145 and PC3 derived from human prostate and in the TRAMP (Transgenic Adenocarcinoma Mouse Prostate) model for prostatic cancer chemoprevention. NAO exerted an antiproliferative effect on DU145 and PC3 cells. Inhibition of cellular proliferation occurred in a dose-dependent manner (IC50 in the range of 2-4 mM), increasing numbers of G1 cells and reducing hydrogen peroxide and peroxide levels. [Nyska, A., Suttie, A., Bakshi, S., Lomnitski, L., Grossman, S., Bergman, M., Ben-Shaul, V., Crocket, P., Haseman, J. K., Moser, G. Toxicol Pathol 2003, 31, 39-51] Spinacea is a rich source of chlorophyll and Chlorophyllin, a water-soluble form of chlorophyll, is interesting for its antimutagenic and anticarcinogenic properties [Negishi, T., Rai, H., Hayatsu, H. Mutat Res 1997, 376, 97-100]. Topical (2 mg) and oral (100 mg/kg) NAO-treatment to hemizygous Tg.AC mice for 5 days/week for 5 week revealed a significant decrease in multiplicity in mouse skin papilloma in the NAO topically treated group. The effect of NAO in the Tg.AC model may be related to its ability to detoxify peroxides and free radicals [Nyska, A., Lomnitski, L., Spalding, J., Dunson, D. B., Goldsworthy, T. L., Grossman, S., Bergman, M., Boorman, G. Toxicol. Let., 2001, 122, 33-44].
DNA polymerase inhibitors could be employed as anticancer chemotherapy agents because they inhibit cell proliferation. Spinacea contained the large amount of sulfoquinovosyl diacylglycerol (SQDG). Fraction containing the major glycolipids in monogalactosyl diacylglycerol, digalactosyl diacylglycerol and sulfoquinovosyl diacylglycerol (SQDG) from dried vegetables inhibited of DNA polymerase α (pol α) in-vitro and also the proliferation of human cancer cells (inhibition of pol α activity may lead to cell growth suppression,). human stomach cancer cells (NUGC-3) and promyelocytic leukemia (HL-60). The inhibitory effect glycolipids fractions of NUGC-3 was almost equal as that of HL-60. The inhibition by the spinacea glycolipids fraction was dependent on dose, and the LD50 value was 66.8-70.1 μg/ml. both for NUGC-3 and HL-60 cell line [Kuriyama, I., Musumi, K., Yonezawa, Y., Takemura, M., Maeda, N., Iijima, H., Hada, T., Yoshida, H., Mizushina, Y., J Nutr Biochem 2005, 16, 594-601; Maeda, N., Hada, T., Murakami-Nakai, C., Kuriyama, I., Ichikawa, H., Fukumori, Y., Hiratsuka, J., Yoshida, H., Sakaguchi, K., Mizushina, Y. J Nutr Biochem 2005, 16, 121-128].
Antioxidant effects associated with spinacea have a protective effects on neurodegenerative disorders such as ischemia and aging by scavenging reactive oxygen species, generated during cerebral ischemia and reperfusion. Adult male Sprague-Dawley rats were fed with spinacea extract with control diet for 4 weeks had reduced cerebral infarction after ischemia and reperfusion. [Wang, Y., Chang, C.-F., Chou, J., Chen, H.-L., Deng, X., Harvey, B. K., Cadet, J. L., Bickford, P. C. Exp. Neurol. 2005, 193, 75.]. Moreover, supplementation of diet with spinacea improve learning and memory in aged animals and possibly in humans and reverse age-induced dwindle in beta-adrenergic receptor function in cerebellar Purkinje neurons [Bickford, P. C., Gould, T., Briederick, L., Chadman, K., Pollock, A., Young, D., Shukitt-Hale, B., Joseph, J. Brain Res 2000, 866, 211-217; Joseph, J. A., Shukitt-Hale, B., Denisova, N. A., Bielinski, D., Martin, A., McEwen, J. J., Bickford, P. C. J Neurosci 1999, 19, 8114-8121].
Spinacea extract a dose of 400 mg/kg body weight decreased significantly the blood glucose and increased significantly insulin concentrations comparable to standard drug, chlorpropamide [Karami Bonari, A. R. Clinical Biochemistry 2011, 44, S331]. Moreover, α-glucosidase inhibitory activity, insulin like action, insulin sensitizing activity exhibited by whole juice and ethanolic extract. Whole juice samples of spinacea showed 5.2% α-glucosidase inhibition at 50 of the juice solution [Park, J.-H., Kim, R.-Y., Park, E. Food Sci. Biotechnol. 2012, 21, 1187-1193.] Ethanolic extract of Spinacia oleracia leaves in alloxan induced diabetic rat showed 54% decrease in serum glucose level compared to control at 100 mg/kg [Kumar, N. J., Loganathan, Glob. J. Biotech.Biochem. 2010, 5, 87-91], additionally hypoglycaemic activity evaluation of ethanolic and aqueous extract of (200 & 400 mg\kg). by Gomathi et al. resulted in a significant decrease in the elevated blood glucose levels as compared to the control. Studies showed regeneration of β-cells of pancreas improvement in body weight and serum lipid profile [V. Gomathi, B. Jayakar, R. Kothai, G. Ramakrishnan, Antidiabetic activity of leaves of Spinacia oleracea Linn. in Alloxan-induced diabetic rats. J. Chem. Pharm. Res., 2010, 2, 266-274].
Pure Cell Complex (PCT-233), corresponds to the isolation and stabilization of the photosynthetic machinery of the thylakoid membrane from mesophyll tissue of spinacea leaves, was extracted from spinacea leaves [Purcell M. Procedure for preparing active plant extracts used to trap free radicals, the extracts and compounds and devices containing them. Canadian patent: CA 2293852, 1999) PCT], is able to protect IMR-32 cells (human neuroblastoma) from apoptosis caused by the generation of reactive oxygen species (ROS) and singlet oxygen.
Aqueous extract of leaves is effective in almost all the symptoms of inflammatory bowel disease (chronic idiopathic inflammatory intestinal condition characterized by intestinal inflammation and mucosal damage). Treatment with aqueous extract of leaves (500 and 1,000 mg/kg/day) to acetic acid and ethanol induced colitis in mice showed significant increase in body weight, decrease in diarrhea with bloody stool, and increase in hemoglobin in inflammatory bowel disease [Otari, K. V., Gaikwad, P. S., Shete, R. V., Upasani, C. D. Inflammopharmacology 2012, 20, 277-287].
Natural antioxidant mixture (NAO) isolated from spinacea exhibited cardio-protection against Doxorubicin (DOX) induced cardiotoxicity. BALB/C mice were treated with NAO (10 mg/kg, ip) for seven consecutive days before and/or 6 days after DOX administration. NAO at the dose of 130 mg/kg conferred significant cardiac protection [Breitbart, E., Lomnitski, L., Nyska, A., Malik, Z., Bergman, M., Sofer, Y., Haseman, J. K., Grossman, S. Hum Exp Toxicol 2001, 20, 337-345] The methanolic extract fresh plant leaves of Spinacia oleracea was found to be highly active against UTI causing bacteria such as Escherichia coli, Enterococcus faecalis, Klebsiella pneumonia to cure urinary tract infection [Das, M. P., Chatterjee, S. Int. J. Pharm. Sci. Rev. Res., 23, 2013, 23, 211-215].